The present invention relates to an apparatus for reading a luminescence pattern that scans a faint luminescence pattern emitted from a planar sample by scanning it with a rotary disk.
Gel electrophoresis analysis methods have hitherto been widely used for the differentiation and structural analysis of biological macromolecules such as proteins and nucleic acids. In the gel electrophoresis, they are analyzed by exploiting the principle that the migration distance of a sample in electrophoresis differs according to its molecular weight. This analysis method is appropriate for the analysis of minute sample quantities, and in general the sample amounts that can be obtained using gel electrophoresis are therefore often limited. In such cases there is a particular requirement for reliability and high sensitivity in the analysis process.
Reliability is an important concern when only a small amount of sample is available, and so electrophoresis patterns have hitherto been read by using a radioisotope to label the sample to be analyzed before it is injected into the gel where electrophoresis is performed, after which the gel is pasted to an X-ray film or the like which is thereby exposed, and this X-ray film is developed to bring out the pattern exposed by the radioisotope, which is then read.
However, radioisotopes are dangerous and must be handled with extreme care. In recent years, therefore, highly sensitive detection methods employing fluorescence and chemical luminescence (fluorescence method, chemiluminescence method) have been developed. These methods are also used in DNA base sequencing and in various types of tests such as southern plotting, western plotting and northern plotting.
The fluorescence method determines a sample's electrophoresis pattern by labeling the sample with a fluorescent substance and--once the electrophoresis has finished--irradiating laser light onto it and measuring the intensity distribution of fluorescent light emitted by this fluorescent substance. An example of apparatus for reading a fluorescence pattern is disclosed in Examined Japanese Patent Publication (JP-B) No.H8-3481 (or in the specification of U.S. Pat. No. 5,069,769).
Also, chemiluminescence reading methods that have hitherto been developed include the method described by C. Martin, L. Bresnick, R.-R. Juo, J. C. Voyta and I. Bronstein (Improved Chemiluminescent DNA Sequencing; Bio Techniques 8, pp. 110-113, 1991).
In the chemiluminescence method, the electrophoresis pattern is normally read in by hybridizing an enzyme that undergoes luminescence to a labeled probe or the like after electrophoresis has been completed, whereby the luminescence pattern of the sample to be analyzed is made to emit light in a particular way. The luminescence pattern of the luminescent sample is exposed to film for 10 to 30 minutes or thereabouts by bringing a fast film into close contact with a membrane from which the luminescent sample has been transferred, placing it inside a light-shielding case, and regulating the exposure time according to the luminescence.
It is noted that various types of fast film can be used here, such as the X-ray film used for radioisotopes. After the exposed film has been developed, it can be analyzed by visual pattern analysis or by various types of image processing software using image acquisition apparatus such as a camera or image scanner.
Incidentally, the abovementioned conventional luminescence pattern reading apparatus is dedicated to the analysis of samples by the fluorescence method, since reading luminescence patterns with the chemiluminescence method involves manual intervention, no dedicated reading apparatus has yet been specifically developed for reading luminescence with both the fluorescence method and the chemiluminescence method with a single reading apparatus.
Also, in the luminescence pattern reading apparatus of the chemiluminescence method, the light from a planar luminescence pattern that is faintly luminescent over its entire surface must be read before there is any change in the amount of luminescence. This necessitates the use of an optical sensor that is highly sensitive, in which case it is also necessary to have an optical system with a photoreceptor path and a scanning mechanism that condenses and inputs the faint light at the position of each pixel in the luminescence pattern.
To successively scan each individual pixel in the luminescence pattern while distinguishing their positions, it is necessary to have a mechanism that transports a photoreceptor window in two dimensions to positions corresponding to the pixels. To increase the speed at which the entire plane is scanned, the scanning window must be transported at high speed. With a conventional scanning mechanism where the scanning window is transported in straight lines and the sample has a width of, say, 200 mm, it is limited to about 3 reciprocating motions per second even if the mass of the condenser unit constituting the moving photoreceptor window is reduced to the bare minimum.
Incidentally, even if the transport speed at which the photoreceptor window scans the sample surface is increased, it does not necessarily mean that the reading speed of the overall apparatus can be increased without limit, as there is an upper limit determined by the relationship between the amount of light obtained from the position of each individual pixel in the luminescence pattern of the sample and the photosensitivity of the photosensor that is used.
If the condenser unit constituting the moving photoreceptor window is mechanically scanned at high speed (by a scanning mechanism), the photosensor (photoelectric conversion element) where the light from this condenser unit is introduced and detected has to detect amounts of light emitted very faintly such as fluorescence. Consequently, highly sensitive photosensors such as photomultipliers, cooled CCDs and image intensifiers, for example, are used. In this case, to ensure that the photosensor sensitivity remains stable, it is preferable to adopt a mechanical structure whereby the photosensor is attached at a static position rather than being mounted on a moving structure such as the carrier of the scanning mechanism. Furthermore, it is also preferable to be able to read the luminescence patterns of samples emitting different colors of light by discriminating between these colors.